This application is a continuation-in-part of the national phase filing of U.S. Application Ser. No. 09/937,057 filed on Feb. 26, 2002 which claims priority from PCT/FR00/00676 filed on Mar. 17, 2000. Priority to each of these prior applications is expressly claimed, and the entire disclosure and claims of each of these prior applications is hereby incorporated by reference in its entirety. Priority is also claimed to FR 99/03433, filed Mar. 19, 1999.
This invention is in the field of immunostimulatory molecules that are administered to a patient to help the body's immune system respond to a disease, principally a solid, malignant tumor. Specifically, the present invention relates to compositions and methods of use of stabilized oligonucleotides with antitumor activity.
The effective treatment of cancers remains one of the major challenges of medicine today. The effectiveness of conventional surgical therapies or therapies aimed at cytolysis (chemotherapy and radiotherapy) remains very limited in many cancers. For astrocytomas for example, the treatment of which is based mainly on surgical exeresis, and local cerebral irradiation, the survival median is only 4 to 6 months after surgical exeresis and 8 to 10 months with the combination of surgery and radiotherapy. Supplementary chemotherapy prolongs survival in patients under the age of 60, but very modestly, usually by only about 3 months. Under this triple treatment, the survival median remains less than two years for histological grade III (anaplastic astrocytoma) and less than 1 year for grade IV (glioblastoma). The mortality for these two groups is near 100% (Daumas-Duport C. et al. (1988), Cancer 62(10) pp 2152–65).
Stimulation of the immune system in the treatment of cancers has been attempted through the administration of bacterial extracts (Jaeckle K. A. et al. (1990), J. Clin. Oncol. 8(8) pp 1408–18) or bacterial DNA, in particular that of Mycobacterium bovis (MY-1) (Tokunaga T. et al. (1984), JNCI72 pp 955–62). MY-1 is, however, ineffective in increasing survival in a model of glioma in mice (Nakaichi M. et al (1995), J. Vet. Med. Sci. 57(3) pp 583–5). IL-2 (Herrlinger U. et al. (1996), J. Neurooncol. 27(3) pp 193–203) and, more recently, IL-12 (Kishima H. et al. (1998), Br. J. Cancer 78(4) pp 446–53; Jean W. C. et al (1998), Neurosurgery 42(4) pp 850–6) have also been studied.
Unfortunately, previous attempts using bacterial DNA have had limited effectiveness or unacceptable toxicity and, to date, only the Mycobacterium bovis BCG has resulted in clinical applications, and then only in the limited indication of bladder cancer (Soloway M. S. et al. (1988), Urol. Clin. North Am. 15 pp 661–9).
Oligonucleotides are polymers formed by the combination of purine or pyrimidine bases and sugars, in particular ribonucleotides or deoxyribonucleotides. In the natural form, the linkages joining the individual nucleotides are phosphoesters which are sensitive to the nucleases of the human body. Thus, oligonucleotides have a very short in vivo half-life (of about one minute) when they are injected into humans, which limits their biological effects. To prolong the half-life oligonucleotides can be modified to be resistant to nucleases. Several types of stabilized oligonucleotide have thus been created, such as, phosphorothioates or methylphosphonates (Crooke R. M. (1991), Anti-Cancer Drug Design 6 pp 609–46). The most commonly used are phosphorothioate oligonucleotides.
In some applications, oligonucleotides are designed to be complementary to a known DNA sequence and are termed “anti-sense” due to the specific identity and sequence of the nucleotides relative to a native gene. Antisense application of oligonucleotide technology are very well known. Some oligodeoxynucleotides, and in particular some synthetic oligodeoxynucleotides, have biological effects per se due to the inherent nature of the sequences themselves, outside their conventional antisense properties. The injection of oligonucleotides as therapeutic compounds has been explored for many years, and the development of specific sequences and techniques continues to be an important area of research.
Some oligodeoxynucleotides, independently of any known antisense sequence, are known to stimulate, in vitro and in vivo, the proliferation of B lymphocytes and the activity of natural killer (NK) cells, and induce the secretion by the cells of cytokines such as α-IFN, β-IFN, γ-IFN, IL-6, IL-12 or TNF-α (Yamamoto S. et al (1992), J. Immunol. 148(12) pp 4072–6; Yamamoto T. et al. (1994), Microbiol. Immunol. 38(10), pp 831–6; Yi A. K. et al. (1996), J. Immunol. 157(12) pp 5394–402; Ballas Z. K. et al. (1996), J. Immunol. 157(5) pp 1840–5; Cowdery J. S. et al. (1996), J. Immunol. 156(12) pp 4570–5; Stacey K. J. et al. (1996), J. Immunol. 157(5) pp 2116–22). This set of cytokines directs toward a Th1-type immune response (Chu R. S. et al. (1997), J. Exp. Med. 186(10) pp 1623–31).
One specific group of oligonucleotides that have been the subject of extensive research are characterized by the presence of a “CpG” motif. This terminology indicates that the sequence of the oligonucleotide molecule contains the nucleotides cytosine (C) and guanine (G) with a phosphate backbone and exhibits a characteristic structure and function when administered therapeutically to a patient. The immunostimulatory properties of these oligodeoxynucleotides are in large part dependent on nonmethylated CG motifs (nonmethylated CpG dinucleotides) which are under-represented in mammalian DNA (Kuramoto E. et al. (1992), Jpn. J. Cancer Res., 83 pp 1128–31).
While the authors agree on the fact that the nonmethylated CG motif is essential to the immunostimulatory function, the identity of the other nucleotides in the complete oligonucleotide molecule are also crucial and the two nucleotides adjacent to the CG motif also dictate the immunostimulatory activity. Although numerous sequences have been studied, the data published on the function of the adjacent sequences are contradictory. It should be appreciated that small differences in the nucleotide sequence of the oligonucleotide, and any chemical modifications thereof, can yield dramatic differences in the therapeutic utility in vivo. Also, special injection techniques, formulations, and other therapeutic approaches can be used in combination with the specific sequence of the oligonucleotide to yield important differences in overall therapeutic utility.
Specifically, Krieg A. M. et al. ((1996), Antisense Nucleic Acid Drug Dev. 6(2) pp 133–9) describe a hexameric motif of the type 5′ pur-pur-C-G-pyrimidine-pyrimidine 3′, whereas application EP 468 520 claims a palindromic hexameric motif. International application WO 9855495 shows that not all the hexamers as defined by Krieg et al. 1996 are immunostimulatory, and that octamers, of sequence 5′-purine purine CG pyrimidine pyrimidine CC-3′ (pur-pur-CG-pyr-pyr-CC) or of sequence 5′-purine purine CG pyrimidine pyrimidine CG-3′ (pur-pur-CG-pyr-pyr-CG) should be defined to provide immunostimulatory activity.
Other immunostimulatory oligodeoxynucleotides, some which are and some which are not defined as oligonucleotides having a nonmethylated CG motif, have been described in application EP 855 184 and certain binding sequences for eukaryotic transcription factors such as NFκB or the AP-1 family.
Also, the use of immunostimulatory properties of oligodeoxynucleotides with nonmethylated CG-type motif has been applied to several medical fields:
(1) in the field of vaccination, in combination with the antigen, as an adjuvant for stimulating specifically a Th1-type immune response (Davis H. L. et al. (1998), The Journal of Immunology 160(2) pp 870–6, European Patent Application EP 855 184, PCT Application WO 98/18810, University of Iowa Research Foundation, and PCT Application WO 98/55495);
(2) in the field of allergy, for modulating the immune response (International Applications WO 98/18810 and WO 98/55495); and
(3) in the domain of cancer,                either in combination with a tumor antigen, as an adjuvant of an antitumor vaccine (application EP 855 184; Weiner G. J. et al. (1996), Proc. Natl. Acad. Sci. 94, pp 10833–7; Wooldridge J. E. et al. (1997) Blood 89(8) pp 2994–8),        or alone as antitumor agents (Connell et al. (1999), Proceedings of the American association for Cancer Research 40 pp 299; application EP 468 520; Carpentier A. F. et al. (1999), Cancer Research 59, pp 5429–5432.        
In the latter case, the antitumor activity of only a few sequences, among those described, has been effectively demonstrated:                Weiner G. J. et al. and Wooldridge J. E. et al. (already cited) who use an oligonucleotide comprising a nonmethylated CG motif of sequence SEQ ID NO: 49 5′-TCTCCCAGCGTGCGCCAT-3′, show that this oligonucleotide has no antitumor effect when it is used alone;        Carpentier et al., Tokunaga et al. and Connell et al. (mentioned above), who use a phosphorothiate oligonucleotide of the octameric type (SEQ ID NO: 2 5 ′TGACTGTGAACGTTCGAGATGA3′), a nonstabilized palindromic hexameric oligonucleotide (SEQ ID NO: 50 5′ ACCGATGACGTCGCCGGTGACGGCACCACGACGACGGCCA CGTGCT 3′) and a hexameric phosphorothioate oligonucleotide of the type 5′ purine purine CG pyrimidine pyrimidine 3′, respectively, show antitumor activity.        
Other than the nonmethylated CG motif, the exact nature of the active sequences of these immunostimulatory oligodeoxynucleotides, for producing antitumor activity, is not clearly defined; in particular, the data published on the nature of the sequences adjacent to the nonmethylated CG motif (2 bases in the 5′ direction and 2 bases in the 3′ direction (hexameric motifs) or 4 bases in the 3′ direction (octameric motif)) are contradictory as are the teachings of the significance of the identity of these sequences.
Recent studies reported by Hartmann G. et al. ((2000), The Journal of Immunology 164 pp 1617–24) explain the difficulty in defining the sequence of such oligonucleotides. These data indicate that not all immunostimulatory oligodeoxynucleotides are equivalent and effective for all the contemplated field defined above and that the stimulation of different compartments of the immune system to obtain the desired activity: adjuvant, antiallergic or antitumor activity highly depends on the specific nature and composition of the oligonucleotide sequence.
In addition, the immune mechanisms of tumor rejection are poorly understood and the data for stimulation of the compartments of the immune system, in vitro, as defined above do not make it possible to predict in advance the antitumor effectiveness of a given oligonucleotide, and it is therefore important to test their antitumor activity in vivo.
Furthermore, the toxicity of oligodeoxynucleotides containing a CG-type motif has been reported when used systemically (both intravenous IV) and (intraperitoneal IP) and also must be taken into account for therapeutic applications (See EP 855 184). Consequently, the immunostimulatory oligonucleotides comprising a CG motif of the prior art have varying and random antitumor activities and may be toxic. For this reason, a set of effective, non-toxic immunostimulatory sequences for antitumor use has not been defined.